Our research is focused on the initiation steps of the bacteriophage
Mu DNA transposition reaction. We study this reaction not only as a prototypical
example of a family of genetic rearrangement reaction systems wide-spread among
organisms, but also as an example of highly regulated macromolecular complex
assembly reactions that control many important biological processes.
Critical early steps in Mu transposition are a pair of DNA cleavages and strand
transfers involving the ends of the Mu DNA sequence and a target DNA; these
reactions generate a branched DNA intermediate. These chemical reaction steps
take place within higher order protein-DNA complexes called transpososomes,
the core of which is composed of two Mu-end DNA segments that are synapsed by
a stably bound tetramer of MuA transposase protein . Transpososome assembly
is controlled by a number of cofactors: an enhancer type DNA sequence element
called IAS that overlaps the Mu operator sequence and the Mu repressor that
binds to it, the MuB ATPase protein, the E. coli-encoded HU and IHF proteins,
ATP, and Mg++.
Study of Mu transpososome assembly process and its control by accessory protein and DNA cofactors.
By making use of a simplified transpososome asembly reaction system, we and others have shown that both the Mu end DNA cleavage and the subsequent strand transfer at one Mu DNA end are catalyzed by the MuA monomers that are bound to the partner Mu DNA end within a transpososome (Figure 2); this explains why Mu DNA end synapsis is a prerequisite for the catalytic steps. Roles of the IAS and MuB ATPase in the transpososome assembly process are currently under investigation by making use of a reaction system in which a transpososome is assembled from short Mu end DNA fragments. The assembly process is monitored by non-denaturing agarose gel electrophoresis of the stable complexes. We are currently developing fluorescence based systems to monitor the assembly reaction in real time, including single molecule analysis systems described below.
Figure 1
Study of conformational states of the MuB ATPase and its interaction with DNA and MuA transposase protein.
MuB ATPase controls each of the early steps of Mu DNA transposition: It assists transpososome assembly, is involved in the target DNA site selection, activates the MuA transposase for the strand transfer reaction, and protects transpososome from premature disassembly by ClpX chaperon protein until strand transfer is completed and the transposition intermediate is ready for DNA replication by the host replication proteins. In turn, the functional state of MuB is controlled by the ATPase cycle and by its interaction with MuA. Structural and functional aspects of MuB-DNA complex are currently under investigation using a variety of physical and biochemical techniques.
Stereochemical studies of phosphoryl transfer reactions in
DNA recombination and related processes.
Determination of the stereochemistry of enzymatic phosphoryl transfer reactions has long been recognized as a powerful tool for elucidating the mechanisms of these enzymes. DNA recombination reactions involve phosphoryl transfer reactions, such as phosphodiester hydrolysis and DNA strand transfer. We have used chiral phosphorothioate containing substrate DNA to strudy the stereo-specificity of recombination enzymes, and also to study stereochemical course of these reactions.
Development of fluorescence based single
molecule studies of macromolecular assembly processes in Mu transposition
and HIV DNA integration reactions.
We are currently in the process of developing a ultra-high sensitivity fluorescence microscope system to be used for the study of macromolecular complex assembly/ disassembly processes such as those involved in Mu transposition and the HIV DNA integration reaction. Single Molecule Research Update
Structural studies of recombination proteins.
Efforts are continued toward solving the high-resolution structure of domains of MuA transposase as well as protein-DNA complexes in collaboration with scientists in LCP/NIDDK and at Chicago University. Structural studies of MuB protein have been started in collaboration with scientists in DBB/NIDDK.
Last updated, August 12th, 2002
